Marker for distinguishing ginseng species and method for producing ginseng extract with increased immune activity

Abstract

The present invention relates to a method for producing a ginseng type identification marker and a ginseng extract with increased immune activity. It was confirmed that the extraction yield increased when white ginseng (white ginseng) was mixed with white ginseng (white tail ginseng) and subjected to multiple extraction. It was also confirmed that the white ginseng extract extracted by multiple extraction was effective in increasing T cell proliferation. The ginsenoside content ratio of the white ginseng extract extracted by multiple extraction was also confirmed to be effective in increasing T cell proliferation. Furthermore, an extract obtained by mixing the trunks and roots (fine roots) of Korean ginseng and American ginseng in a 7:3 ratio was analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis. Ten ginsenosides specifically found in Korean ginseng and nine ginsenosides specifically found in American ginseng were identified, and metabolite markers differentially found depending on the origin of ginseng were also identified.

Description

[Technical Field]

[0001] The present invention relates to a marker for distinguishing ginseng varieties and a method for producing a ginseng extract with increased immune activity. [Background technology]

[0002] Immunity is a self-defense system present in the body, which is the process by which the human body recognizes various substances and living organisms that invade from the outside as foreign bodies, removes them, and metabolizes them.

[0003] The immune system protects the body from damage caused by external stimuli and invasion by pathogenic microorganisms, but can also damage self-tissues, such as in inflammatory reactions. The effects of regulating changes in immune function to restore normality or reduce the extent of changes are classified as immune function suppression and immune function enhancement. Alleviating hypersensitive immune responses means suppressing unwanted increased immune responses, such as allergic reactions caused by adverse reactions to external substances, and reactions to self-antigens or modified self-antigens.

[0004] In order to overcome the problem of the occurrence of such immune responses, various immune enhancing agents and therapeutic agents have been used, but they have problems such as side effects, and most of the commercially available agents are taken for treatment rather than prevention. Therefore, there is a need for an immune enhancing composition that can be taken for a long period of time without side effects and is also suitable for prevention.

[0005] Ginseng, also known as Korean ginseng (Panax ginseng CA Meyer), is a perennial herbaceous plant whose roots are primarily used for medicinal purposes in traditional Chinese medicine. Ginseng's chemical and pharmacological properties have revealed that it contains a variety of components, including saponin (ginsenoside), phenolic compounds, polyacetylene, and polysaccharide. Ginseng, in particular, contains large amounts of saponin, a compound known to be effective in regulating biological functions. Ginseng's saponin (ginsenoside) is known to have beneficial effects on immune function, anticancer activity, antioxidant activity, improvement of neurological disorders, and the prevention, amelioration, or treatment of adult diseases such as diabetes and hypertension. Many research results have been reported regarding the physiological activity of ginseng or red ginseng in relation to cancer, and it is known that ginsenoside-Rb2, ginsenoside-Rg3, Rh1, and Rh2 have high activity. In particular, it has recently been reported that non-saponin compounds (polysaccharides and polypeptides) also contribute to regulating immune function and suppressing tumors.

[0006] Furthermore, ginseng is cultivated in large quantities in Korea and is widely used in traditional folk remedies to strengthen the body and boost the immune system. Recently, in Korea, Japan, and China, the saponin component (ginsenoside) extracted from ginseng has been widely used as a pharmaceutical ingredient or a medicinal material. The main component that determines the physiological activity of ginseng is saponin, and its efficacy varies depending on the content and type of saponin. In an attempt to enhance the physiological activity of ginseng, ginseng is heated and fumigated to induce chemical changes and concentration of the saponin component, which not only strengthens physiological activity but also creates new physiological activity.

[0007] Meanwhile, many herbal medicines produced and distributed in Korea are often confused or misused due to inaccurate origins or similar names. Furthermore, countries that use herbal medicines, such as Korea, China, and Japan, have different definitions for the origins of medicinal plants. Korea, which relies on imports for over 80% of its herbs, is therefore directly exposed to such confusion and misuse. Traditional methods for distinguishing between confused or misused herbal medicines and their origins include morphological sensory testing, physicochemical testing, cytological testing, and physiological testing. However, because herbal medicines are natural products, it is difficult to ensure consistent quality. This is because the components of each individual herb vary greatly, and significant differences can occur depending on the breeding method, harvesting time, growing environment, and part of the herb. Recently, one of the herbal medicines that has become the subject of the most controversy in the herbal medicine distribution market due to confusion and misuse related to its origin is Korean ginseng (Panax ginseng CA Meyer) and American ginseng (Panax quinquefolius). Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide an immune-boosting food composition containing a mixed extract of the trunk and rootlets of ginseng (Panax ginseng CA Meyer) as an active ingredient.

[0009] Another object of the present invention is to provide a pharmaceutical composition for enhancing immunity, which comprises a mixed extract of the trunk and rootlets of ginseng (Panax ginseng CA Meyer) as an active ingredient.

[0010] Another object of the present invention is to provide a method for preparing a ginseng mixture by mixing the trunks and rootlets of ginseng (Panax ginseng CA Meyer); preparing a primary extract using the mixed ginseng mixture as a primary solvent; filtering the primary extract and extracting the residue with a secondary solvent to prepare a secondary extract; filtering the secondary extract and extracting the residue with a third solvent to prepare a third extract; and and mixing and concentrating the first, second and third extracts.

[0011] Another object of the present invention is to provide a method for preparing a ginseng mixture by blending the trunks and rootlets of Korean ginseng or American ginseng; Extracting the ginseng mixture to produce an extract; and and analyzing the extract using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

[0012] Another object of the present invention is to provide a method for producing a ginseng extract comprising the steps of extracting Korean ginseng or American ginseng rootlets to produce an extract; and analyzing the extract using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

[0013] It is still another object of the present invention to provide a method for enhancing immunity, comprising administering to an individual a pharmaceutically effective amount of a mixed extract of the trunk and rootlets of ginseng (Panax ginseng CA Meyer). [Means for solving the problem]

[0014] To achieve the above object, the present invention provides an immune-boosting food composition containing a mixed extract of the trunk and rootlets of ginseng (Panax ginseng CA Meyer) as an active ingredient.

[0015] The present invention also provides a pharmaceutical composition for enhancing immunity, which comprises a mixed extract of the trunk and rootlets of ginseng (Panax ginseng CA Meyer) as an active ingredient.

[0016] The present invention also provides a method for preparing a ginseng mixture by mixing the trunks and rootlets of ginseng (Panax ginseng CA Meyer); preparing a primary extract using the mixed ginseng mixture as a primary solvent; filtering the primary extract and extracting the residue with a secondary solvent to prepare a secondary extract; filtering the secondary extract and extracting the residue with a third solvent to prepare a third extract; and and mixing and concentrating the first extract, the second extract, and the third extract.

[0017] The present invention also provides a method for preparing a ginseng mixture by mixing the trunks and rootlets of Korean ginseng or American ginseng; Extracting the ginseng mixture to produce an extract; and and analyzing the extract using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

[0018] The present invention also provides a method for producing a ginseng extract, comprising the steps of extracting Korean ginseng or American ginseng rootlets to produce an extract; and analyzing the extract using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

[0019] The present invention also provides a method for enhancing immunity, comprising administering to an individual a pharmaceutically effective amount of a mixed extract of the trunk and rootlets of ginseng (Panax ginseng CA Meyer). [Effects of the Invention]

[0020] The present invention confirmed that the extraction yield increases when white ginseng (white ginseng) is mixed with white tail ginseng and subjected to multiple extraction. It also confirmed that the white ginseng extract extracted by multiple extraction is effective in increasing T cell proliferation. It also confirmed that the ginsenoside content ratio of the white ginseng extract extracted by multiple extraction is excellent in T cell proliferation. Furthermore, extracts extracted by mixing the trunks and roots (fine roots) of Korean ginseng and American ginseng in a 7:3 ratio were analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis. Ten ginsenosides specifically contained in Korean ginseng and nine ginsenosides specifically contained in American ginseng were identified. This identification of metabolite markers differentially contained depending on the origin of ginseng can be useful in related industries. [Brief explanation of the drawings]

[0021] [Figure 1] 1 is a quantification of the yield of ginseng extract extracted by the multiple extraction method of the present invention. [Figure 2] 1 shows the quantification of the T cell proliferation effect of the ginseng extract extracted by the multiple extraction method of the present invention. [Figure 3] 1 shows the quantification of the ginsenoside contents of ginseng extract and American ginseng extract extracted by the multiple extraction method of the present invention. [Figure 4] FIG. 1 is a graph comparing the T cell proliferation effects of different extraction methods and ginsenoside content ratios. [Figure 5]FIG. 1 is a diagram showing the analysis of ginsenosides in the ginseng extract of the present invention by UPLC analysis. [Figure 6] FIG. 1 shows the results of UPLC analysis of ginsenosides in American ginseng extract. [Figure 7] The metabolites of Korean ginseng extract and American ginseng extract were analyzed by principal component analysis (PCA) based on UPLC-QTOP / MS, and the score plots of PC1 and PC2 are shown. [Figure 8] The metabolites of Korean ginseng extract and American ginseng extract were analyzed by orthogonal partial least squares discriminant analysis (OPLS-DA) based on UPLC-QTOP / MS. [Figure 9] 1 shows the score plots of metabolites of Korean ginseng extract and American ginseng extract analyzed by OPLS-DA based on UPLC-QTOP / MS. [Figure 10] FIG. 1 shows the analysis of metabolites from Korean ginseng root extract and American ginseng root extract by OPLS-DA based on UPLC-QTOP / MS. [Figure 11] 1 shows the score plots of metabolites from Korean ginseng root extract and American ginseng root extract analyzed by OPLS-DA based on UPLC-QTOP / MS. DETAILED DESCRIPTION OF THE INVENTION

[0022] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of well-known technologies known to those skilled in the art may be omitted. Furthermore, in the description of the present invention, if it is determined that a detailed description of related well-known functions or configurations may unnecessarily obscure the gist of the present invention, such detailed description may be omitted. Furthermore, the terminology used in this specification is used to appropriately express preferred embodiments of the present invention, and may vary depending on the intentions of users or operators, the practices in the field to which the present invention pertains, etc.

[0023] Therefore, the definition of this term should be based on the entire contents of this specification. Throughout this specification, when a part "comprises" a certain component, this does not mean that other components are excluded, but that other components may also be included, unless otherwise specified.

[0024] The present invention provides an immunity-boosting food composition containing a mixed extract of the trunk and rootlets of ginseng (Panax ginseng CA Meyer) as an active ingredient.

[0025] The term "prevention" as used in the present invention means any action of suppressing the symptoms of a specific disease or delaying its progression by administering the composition of the present invention.

[0026] The term "amelioration" as used herein means any action that at least reduces the parameters associated with the condition being treated, for example the severity of the symptoms.

[0027] The food composition of the present invention may contain, in addition to the active ingredient of the present invention, various flavoring agents or natural carbohydrates as additional ingredients, as in conventional food compositions.

[0028] Examples of the natural desalinated substances include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The flavoring agents used may be natural flavorings (thaumatin), stevia extracts (e.g., rebaudioside A and glycyrrhizin), and synthetic flavorings (saccharin and aspartame). The food compositions of the present invention can be formulated in the same manner as the pharmaceutical compositions and used as functional foods or added to various foods. Examples of foods to which the compositions of the present invention can be added include beverages, meats, chocolates, snacks, pizza, ramen, other noodles, gums, candies, ice cream, alcoholic beverages, vitamin complexes, and dietary supplements.

[0029] In addition to the extract as an active ingredient, the food composition may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. The food composition of the present invention may also contain fruit pulp for producing natural fruit juices, fruit juice beverages, and vegetable beverages.

[0030] The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of enhancing immunity. In this invention, the term "functional health food composition" refers to a food manufactured and processed using raw materials or ingredients with functional properties beneficial to the human body as defined in Act No. 6727 on Functional Health Foods, and is intended to be consumed for the purpose of obtaining beneficial health effects, such as regulating nutrients or exerting physiological effects on the structure and function of the human body. The functional health food of the present invention may contain conventional food additives, and unless otherwise specified, the suitability of a food additive is determined in accordance with the specifications and standards for that item in accordance with the General Provisions and General Test Methods for Food Additives approved by the Korea Food and Drug Administration. Examples of items listed in the "Food Additives Revolution" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamon; natural additives such as glycerin, licorice extract, crystalline cellulose, high-dose coloring, and guar gum; and mixed preparations such as monosodium L-glutamate preparations, noodle-additive alkalizing agents, preservative preparations, and tar color preparations. For example, health functional foods in tablet form can be prepared by granulating a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, and other additives using conventional methods, adding lubricants, and then compressing the mixture, or by directly compressing the mixture. Furthermore, health functional foods in tablet form can also contain flavoring agents, etc., as needed. Among health functional foods in capsule form, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with excipients and other additives into a conventional hard capsule, while soft capsules can be prepared by filling a mixture of the active ingredient of the present invention with excipients and other additives into a capsule base such as gelatin. The soft capsule may optionally contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, etc. The health functional food in pill form may be prepared by molding a mixture of the active ingredient of the present invention with an excipient, a binder, a disintegrant, etc., using a known method, and may be coated with white sugar or other coating agents, or may be coated with a substance such as starch or talc, if necessary.The health functional food in granular form can be manufactured into granules by mixing the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a known method, and can contain flavoring agents, taste masking agents, etc. as needed.

[0031] According to one embodiment of the present invention, the ginseng trunk may be a ginseng skin directly, and the ginseng skin directly may be a dried ginseng original.

[0032] The "skin-dried ginseng" of the present invention refers to ginseng that has been sun-dried or hot-air-dried without peeling, and is also called white ginseng, and refers to ginseng that has been processed to have a moisture content of 15% or less and has maintained its original shape.

[0033] According to one embodiment of the present invention, the fine root may be White-tailed Panax notoginseng.

[0034] The "white tail ginseng" of the present invention refers to the fine roots (fiber roots) that are a by-product of ginseng and are dried in their original form without any processing.

[0035] According to one embodiment of the present invention, the ginseng trunk and rootlets may be mixed in a weight ratio of 3-9:1-7, preferably 4-8:2-6, and more preferably 5:5, 6:4, and 7:3, but are not limited thereto.

[0036] According to one embodiment of the present invention, the mixed extract may be extracted using a solvent selected from the group consisting of water, C1-C4 lower alcohols, lower alcohol aqueous solutions, hexane, chloroform, and acetone, preferably water or lower alcohol aqueous solutions, but is not limited thereto.

[0037] According to one embodiment of the present invention, the mixed extract may be obtained by filtering the residue after extraction, and the residue may be further extracted with a solvent, or the mixed extract may be obtained by mixing all of the filtrates obtained by the additional extraction.

[0038] According to one embodiment of the present invention, the immune enhancement may be an increase in proliferation of immune cells, and the immune cells may be T cells.

[0039] According to one embodiment of the present invention, the mixed extract may contain ginsenosides Rg1, Rf and Rb, and the ginsenosides Rg1, Rf and Rb may be contained in a weight ratio of 8-10:4-6:18.5-22.5, preferably 9:4:19 or 9:5:22, but are not limited thereto.

[0040] The weight ratio of Rg1, Rf and Rb of the present invention refers to the content of Rg1, Rf and Rb contained in the ginseng multiple extract, and if the weight ratio (content ratio) of Rg1, Rf and Rb is 9:4:19 or 9:5:22, it can increase T cell proliferation compared to the baseline value of the control group.

[0041] The present invention also provides a pharmaceutical composition for enhancing immunity, which comprises a mixed extract of the trunk and rootlets of ginseng (Panax ginseng CA Meyer) as an active ingredient.

[0042] The term "treatment" as used herein means any action that improves or beneficially alters the symptoms of a particular disease by administering a composition of the present invention.

[0043] The pharmaceutical composition of the present invention may further contain an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation, and for example, Freund's complete or incomplete adjuvant may be further included to enhance its effectiveness.

[0044] The pharmaceutical composition according to the present invention may be prepared by mixing the active ingredient with a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0045] The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods.

[0046] When formulated, they can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid formulations can be prepared by mixing the active ingredient with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, syrups, and the like. In addition to commonly used diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Examples of non-aqueous solvents and suspending agents that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of suppository bases that can be used include witepsol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0047] The pharmaceutical composition according to the present invention can be administered to an individual by various routes, including, for example, oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection, although any mode of administration is contemplated.

[0048] The dosage of the pharmaceutical composition according to the present invention is selected taking into consideration the age, weight, sex, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient contained in the pharmaceutical composition can be selected in various ways depending on the subject, and preferably it is contained in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, the pharmaceutical activity may not be exhibited, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0049] The present invention also provides a method for preparing a ginseng mixture by mixing the trunks and rootlets of ginseng (Panax ginseng CA Meyer); preparing a primary extract using the mixed ginseng mixture as a primary solvent; filtering the primary extract and extracting the residue with a secondary solvent to prepare a secondary extract; Filtration of the second extract and extraction of the residue with a third solvent to prepare a third extract; and and mixing and concentrating the first extract, the second extract, and the third extract.

[0050] The present invention also provides a method for preparing a ginseng mixture by mixing the trunks and rootlets of Korean ginseng or American ginseng; Extracting the ginseng mixture to produce an extract; and and analyzing the extract using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

[0051] The term "ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometer (UPLC-QTOF / MS)" used in the present invention refers to a combination of an ultra performance liquid chromatography (UPLC, Ultra Performance Liquid Chromatography) device and a quadrupole time-of-flight mass spectrometer (QTOF / MS, Quadrupole time-of-flight Mass Spectrometer) device.

[0052] The term "gradient elution" used in the present invention refers to a method of elution (column liquid chromatography) or development (thin-layer chromatography) while continuously changing the eluent (composition) of the mobile phase, and is also called gradient elution, inclined gradient elution, or gradient elution.

[0053] The term "multivariate statistical analysis" used in this invention refers to a statistical analysis method used to analyze the characteristics of data containing many interrelated variables (multivariates) and to express their overall characteristics, and analyzes two or more variables. Examples include factor analysis, principal component analysis, and cluster analysis.

[0054] The term "principle component analysis (PCA)" used in the present invention is a statistical method that explains the variation among multiple variables in terms of a mixture of a small number of independent factors called principal components.

[0055] The term "orthogonal partial least squares discriminant analysis (OPLS-DA)" used in this invention refers to an analytical method that rotates axes to derive latent variables that contribute to class division, and then derives biomarkers, which are the most significant variables separated. OPLS-DA analysis is designed to handle deviations of X perpendicular to Y.

[0056] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer, liquid chromatography can be performed using 0.1% formic acid in water (v / v) and 0.1% formic acid in acetonitrile (v / v) as mobile phases and gradient elution.

[0057] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer, mass analysis may be performed using an anion electrospray method.

[0058] According to one embodiment of the present invention, the origin of the Korean ginseng and American ginseng can be determined by analyzing the major metabolites of the sample using an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer (UPLC-QTOF / MS) and performing multivariate statistical analysis on the types and amounts of the metabolites.

[0059] According to one embodiment of the present invention, the multivariate statistical analysis method may be a multivariate statistical analysis method using data from principal component analysis (PCA) or orthogonal partial least squares discriminant analysis (OPLS-DA).

[0060] According to one embodiment of the present invention, the ginseng may contain a metabolite selected from the group consisting of ginsenoside Rf, Rb2, malonyl-Rb2, Rg1, malonyl-Rc, 20-O-glucoginsenoside Rf, Rg2 and Rc.

[0061] According to one embodiment of the present invention, the American ginseng may contain a metabolite selected from the group consisting of ginsenoside XVII, Re, malonyl-Rb1, pseudo-ginsenoside F11 and Rb1.

[0062] The present invention also provides a method for producing a ginseng extract, comprising the steps of extracting Korean ginseng or American ginseng rootlets to produce an extract; and analyzing the extract using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

[0063] According to one embodiment of the present invention, the ginseng may contain a metabolite selected from the group consisting of 20-O-glucoginsenoside Rf, Ra2, Ra1, Rf, notoginsenoside R2, Rh1 and Rg1.

[0064] According to one embodiment of the present invention, the American ginseng may contain a metabolite selected from the group consisting of vinaginsenoside R4, notoginsenoside R1, notoginsenoside Fe, Re, compound O, and pseudo-ginsenoside F11 and F2.

[0065] The present invention also provides a method for enhancing immunity, comprising administering to an individual a pharmaceutically effective amount of a mixed extract of the trunk and rootlets of ginseng (Panax ginseng CA Meyer).

[0066] The immune-enhancing method of the present invention involves administering a therapeutically effective amount of the ginseng trunk and rootlet extract to an individual. The specific therapeutically effective amount for a particular individual will vary depending on various factors, including the type and degree of response to be achieved, the specific composition (including whether other preparations are used), the individual's age, weight, general health, sex, and diet, the administration time, administration route, composition secretion rate, treatment duration, and drugs used in conjunction with or concurrently with the specific composition, as well as similar factors well known in the pharmaceutical arts. The daily dosage is 0.0001 to 100 mg / kg, preferably 0.01 to 100 mg / kg, based on the amount of the pharmaceutical composition of the present invention, and may be administered 1 to 6 times daily. However, it is obvious to those skilled in the art that the dose or administration amount of each active ingredient must be within a range that does not result in side effects due to excessively high concentrations of each active ingredient. Therefore, the effective amount of the composition suitable for the purpose of the present invention is preferably determined taking into account the above factors.

[0067] The individual can be any mammal, including humans and primates, as well as domestic animals such as cows, pigs, sheep, horses, dogs, and cats.

[0068] The active ingredient of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes, including oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection. [Example]

[0069] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of more specifically illustrating the present invention, and that the scope of the present invention is not limited to these examples.

[0070] <Example 1> Preparation of ginseng extract using multiple extraction method In the present invention, a ginseng extract with enhanced immune-enhancing activity and capable of identifying the origin of ginseng was extracted using multiple extraction methods. Specifically, a white ginseng mixture was prepared by mixing the dermal and white tail ginseng of Korean ginseng (Panax ginseng CA Meyer) in weight ratios of 5:5, 6:4, and 7:3, respectively. For the primary extraction, 60% alcohol was added in an amount 8 times the weight of the white ginseng mixture, and the mixture was refluxed at 70-90°C for 8 hours. The extract was then filtered, and the residue was added to 30% alcohol in the same solvent ratio as above, and the secondary extraction was performed at 70-90°C for 8 hours. For the tertiary extraction, the extract was filtered, and the remaining residue was immersed in distilled water in the same ratio as above, and then refluxed in hot water at 70-90°C for 8 hours. All the extracts obtained (filtrates from the primary, secondary, and tertiary extracts) were then mixed, concentrated under reduced pressure, and freeze-dried to produce a multi-ext. ginseng extract. As a control, a white ginseng mixture was refluxed with 50% alcohol (50% ext.) or 70% alcohol (70% ext.) under the same temperature and time conditions as above. The sample names, abbreviations, and extraction yields of the ginseng extracts of the present invention are shown in Table 1 and Figure 1.

[0071] In addition, to confirm whether it is possible to distinguish the origin of American ginseng (Panax quinquefolius) from that of white ginseng (Panax ginseng CA Meyer) and white tail ginseng, respectively, were mixed in weight ratios of 5:5, 6:4, and 7:3, and then refluxed and extracted with 70% alcohol.

[0072] [Table 1]

[0073] As a result, as shown in Figure 1, it was confirmed that the yield of the ginseng extract extracted by the multiple extraction method was significantly higher than that of the 50% or 70% alcohol extract.

[0074] <Example 2> Confirmation of immune-boosting effect of white ginseng extract The immune-enhancing effect of ginseng extract extracted by the multiple extraction method of the present invention was confirmed. Specifically, mice were humanely sacrificed and spleen tissue was obtained. The obtained spleen tissue was pulverized with a sterile glass rod to release the cells. The isolated cell suspension was then passed through a 200-mesh stainless steel sieve in RPMI 1640 medium (Hyclone, Logan, UT, USA) to release the cells. The isolated cell suspension was then passed through 100 μm cell strainers (BD Falcon, Palo Alto, CA, USA), washed twice with culture medium, centrifuged at 3,000 rpm for 10 minutes, and then treated with red blood cell lysing buffer (Hybri-Max™, Sigma-Aldrich) for 5 minutes to remove red blood cells. The erythrocyte-free splenocytes were dispersed again in RPMI 1640 culture medium, stained with trypan blue solution, and counted using a hemocytometer. The erythrocyte-free splenocytes were then placed in a 96-well plate at 1x10 6After distributing the cells / well, each group was treated with the extracts from Example 1 at a concentration of 100 or 200 μg / ml. After 24 hours of incubation in a 37°C, 5% CO2 incubator, 10 μL of WST-1® (Daeil Lap Science, Seoul, Korea) solution was added to each well and incubated for 2 hours. The absorbance was then measured at 450 nm. An untreated control group was used, and Con A (concanavalin A, 2 μg / mL), which induces T cell proliferation, was used as a positive control. The measured absorbance values were used to quantify the proliferation potential of splenocytes using the following equation:

[0075]

number

[0076] As a result, as shown in Figure 2, T cell proliferation was increased in the 50% ext. and 70% ext. groups compared to the control group, but was significantly lower than in the positive control group, Con A. However, the Multi-ext. group, which used the multiple extraction method, showed a concentration-dependent increase in T cell proliferation, and in particular, at a concentration of 200 μg / ml, there was no significant difference from the positive control group, Con A, confirming its superior T cell proliferation ability.

[0077] <Example 3> Comparison of ginsenoside content and immune-boosting effects of ginseng according to its origin <3-1> Comparison of ginsenoside content by origin We investigated whether the index components of white ginseng extract extracted by the multiple extraction method of the present invention differ from those of American ginseng depending on the extraction method. Specifically, the contents of ginsenosides Rg1, Rf, and Rb1 in the ginseng multiple extracts (7:3 and 6:4 multi-extracts) of Example 1, the 70% ginseng blend alcohol extracts (7:3 and 6:4), and the 70% American ginseng blend alcohol extracts (7:3 and 6:4) were analyzed by high performance liquid chromatography (HPLC). The results are shown in Table 2 and Figure 3.

[0078] [Table 2]

[0079] As a result, as shown in Figure 3, Rf was detected in all of the ginseng compared to American ginseng.

[0080] <3-2> Confirmation of immune-boosting effects based on ginsenoside content ratios by origin The immune-enhancing effect of white ginseng extracts extracted by the multiple extraction method of the present invention was confirmed based on the content ratio of indicator components. Specifically, T cell proliferation within spleen cells was evaluated using the same method as in Example 2. In Example 3-1, the content ratios of the indicator components Rg1, Rf, and Rb1 in the extracts were all different, so the immune cell proliferation ability was evaluated based on each ratio. The contents of Rg1, Rf, and Rb1 in each extract are shown in Table 3 below, and the immune-enhancing effect of each extract at a concentration of 50 μg / ml was compared.

[0081] [Table 3]

[0082] As a result, as shown in Figure 4, the American ginseng extract group, with Rg1:Rb1 content ratios of 1:22 and 1:23, showed significantly lower T cell proliferation activity compared to the positive control group, Con A. However, the Korean ginseng extract group showed significantly higher T cell proliferation activity compared to the American ginseng extract group. In particular, the Korean ginseng multiple extract group, with Rg1:Rf:Rb1 content ratios of 9:4:19 and 9:5:22, showed no significant difference in T cell proliferation activity compared to the positive control group, confirming that the above-mentioned ratios of the index components are superior in T cell proliferation activity.

[0083] <Example 4> Preparation for confirming metabolite markers for identifying the origin of Korean ginseng and American ginseng <4-1> Preparation of samples for identifying the origin of Korean ginseng and American ginseng To identify metabolite markers that can distinguish the origin of ginseng and American ginseng in the extracts of the present invention's ginseng or American ginseng mixture, samples were prepared for UPLC-QTOF / MS. Specifically, ginseng trunks and roots were mixed in a 7:3 ratio, and the resulting ginseng mixture and an American ginseng mixture prepared in the same manner were ground in a blender. 50 mg of each sample was used for metabolite extraction. 10 ml of 70% methanol was added to each ground sample, and ultrasonic extraction was performed at room temperature for 60 minutes. Each extract was then filtered to remove impurities, and the final analytical concentration was standardized to 5,000 ppm. The extracts were then used for ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS).

[0084] In addition, fine roots (fiber roots) of Korean ginseng and American ginseng were extracted using the same method as above and used for UPLC-QTOF / MS analysis.

[0085] <4-2>UPLC analysis UPLC analysis was performed using a Waters ACQUITY UPLC system (Waters Corp., MA, USA). A Thermo Hypersil Gold (2.1 x 100 mm, 1.9 μm) column was used, and the mobile phase consisted of water (A) supplemented with 0.1% formic acid and acetonitrile (B) supplemented with 0.1% formic acid. The column oven was maintained at 40°C, and the mobile phase consisted of solvent A (0.1% formic acid in water, v / v) and solvent B (0.1% formic acid in acetonitrile, v / v). The optimal LC elution conditions were as follows: 0–0.5 min, 15% B; 0.5–1 min, 15–20% B; 1–6 min, 20–20% B; 6–13 min, 20–30% B; 13–23 min, 30–35% B; 23–24 min, 35–38% B; 24–27 min, 38–60% B; and 27–31 min, 60–90% B. The flow rate was 0.5 mL / min. 2 μL aliquots were injected onto the column using an autosampler.

[0086] <4-3>Establishment of Q-TOF / MS conditions Q-TOF / MS analysis was performed using a Q-TOF Micromass Detector (Waters, Manchester, UK) to establish optimal conditions for analyzing ginseng and American ginseng metabolites in negative and positive modes. Comparisons revealed that negative mode electrospray effectively analyzed a variety of metabolites. The analytical conditions were as follows: capillary voltage 2.3 kV, cone voltage 40 V, low energy 6 V, high energy 20-45 V, desolvation temperature 550°C, source temperature 120°C, cone gas flow 30 L / h, and desolvation gas flow 800 L / h. Precursor ion data were collected between m / z 100 and 2,000.

[0087] <Example 5> Confirmation of metabolite markers for identifying the origin of Korean ginseng and American ginseng <5-1> UPLC-QTOF / MS results UPLC-QTOF / MS spectra were recorded three times for 12 samples each of Korean ginseng and American ginseng, each pooled into 3-5 portions. UPLC coupled with QTOF / MS, which allows accurate mass measurement, was performed for 30 minutes for various metabolites. Under optimal analytical conditions, Korean ginseng and American ginseng metabolites showed excellent separation. Furthermore, isosolvent elution did not provide clear separation due to the similar polarity and structure of the metabolites, whereas gradient elution provided better separation. Electrospray ionization was tested in both anion and cation modes, with the cation mode being more sensitive for all components. Individual ginsenoside peaks could be identified in the total ion chromatogram by matching molecular weights or fragment ions in the mass spectrum.

[0088] The results of analyzing Korean ginseng and American ginseng using the UPLC and Q-TOF / MS conditions described above are shown in Figures 5 and 6, respectively. Figures 5 and 6 show the UPLC and Q-TOF / MS chromatograms of one representative sample of Korean ginseng and American ginseng. Each chromatogram was observed from 0.1 to 30.0 minutes. The y-axis of the chromatogram (BPI counters) indicates the amount of ion value of each individual compound. This chromatogram sample was used for subsequent multivariate statistical analysis.

[0089] <5-2> Multivariate statistical analysis based on UPLC-QTOF / MS data - Principal component analysis (PCA) The metabolite profile information obtained from the UPLC-QTOF / MS results for Korean ginseng and American ginseng was submitted to PCA analysis, which performs statistical analysis of the two sample groups without any information on the differences between the samples. Starting from the Y-axis, it was confirmed that the left side was Korean ginseng and the right side was American ginseng (Figure 7).

[0090] <5-3> Multivariate statistical analysis based on UPLC-QTOF / MS data - S-Plot based on orthogonal partial least squares discriminant analysis (OPLS-DA) The metabolite profile information obtained from the UPLC-QTOF / MS analysis of Korean ginseng and American ginseng was subjected to OPLS-DA analysis, which maximized the two sample groups and performed statistical analysis. OPLS-DA was performed using SIMCA P15 (Umetrics, Umea, Sweden). An S-plot was then generated based on the OPLS-DA analysis. The S-plot provides information on whether a signal determines the classification of groups in the OPLS-DA analysis method, and the S-plot is displayed for all mass values (m / z).

[0091] As a result, as shown in Figure 8, it was confirmed that the OPLS-DA analysis accurately distinguished between ginseng on the left side of the y-axis and American ginseng on the right side.

[0092] Furthermore, as a result of the S-Plot analysis using the OPLS-DA analysis platform, as shown in Figure 9, the representative mass values displayed below the x-axis indicate markers characteristic of the Korean ginseng sample, and the representative mass values displayed above indicate markers characteristic of the American ginseng sample. This indicates that these are the compounds that contribute most to distinguishing OPLS-DA.

[0093] <5-4> UPLC-QTOF / MS data-based OPLS-DA and S-Plot analysis of root extracts Based on the UPLC-QTOF / MS data of the root extracts of Korean ginseng and American ginseng, OPLS-DA and S-Plot analysis were performed in the same manner as in Example 5-3.

[0094] As a result, as shown in FIG. 10, it was confirmed that, starting from the y-axis of OPLS-DA, the left side was accurately identified as Korean ginseng and the right side as American ginseng.

[0095] In addition, based on the OPLS-DA analysis, S-Plot analysis was performed, and it was confirmed that, starting from the x-axis, the representative mass values displayed below represent markers characteristic of the Korean ginseng sample, and the representative mass values displayed above represent markers characteristic of the American ginseng sample (Figure 11).

[0096] Therefore, the present invention confirmed that the extraction yield increases when white ginseng and white tail ginseng are mixed and extracted multiple times. It was also confirmed that the white ginseng extract extracted by multiple extraction is effective in increasing T cell proliferation, and that the ginsenoside content ratio of the white ginseng extract extracted by multiple extraction is excellent for T cell proliferation. Furthermore, in an extract obtained by mixing the trunks and roots (fine roots) of Korean ginseng and American ginseng in a 7:3 ratio, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis were used to identify 10 ginsenosides specifically contained in Korean ginseng and 9 ginsenosides specifically contained in American ginseng, and to identify metabolite markers that are differentially contained depending on the origin of ginseng.

Claims

1. An immune-boosting food composition comprising a mixed extract of the trunk and rootlets of ginseng (Panax ginseng C.A. Meyer) as an active ingredient.

2. The food composition according to claim 1, wherein the carrot body is directly connected to the skin.

3. The food composition according to claim 2, wherein the ginseng extract is dried ginseng extract.

4. The food composition according to claim 1, wherein the rootlets are those of Shirao-san.

5. 2. The food composition according to claim 1, wherein the carrot body and rootlets are mixed in a weight ratio of 3-9:1-7.

6. The food composition according to claim 1, wherein the mixed extract is extracted using a solvent selected from the group consisting of water, C1 to C4 lower alcohols, lower alcohol aqueous solutions, hexane, chloroform, and acetone.

7. The food composition according to claim 6, wherein the mixed extract is obtained by filtering the residue after extraction.

8. The food composition of claim 7, wherein the residue is further extracted with a solvent.

9. The food composition according to claim 1, wherein the immune enhancement is an increase in the proliferation of immune cells.

10. The food composition of claim 9 , wherein the immune cells are T cells.

11. The food composition according to claim 1, wherein the mixed extract contains ginsenosides Rg1, Rf and Rb.

12. The food composition according to claim 11, wherein the ginsenosides Rg1, Rf and Rb are contained in a weight ratio of 8-10:4-6:18.5-22.

5.

13. The food composition according to claim 12, wherein the weight ratio of Rg1, Rf, and Rb is 8-10:4-6:18.5-22.5, which increases T cell proliferation compared to the baseline value of a control group.

14. A pharmaceutical composition for enhancing immunity, comprising a mixed extract of the trunk and rootlets of ginseng (Panax ginseng CA Meyer) as an active ingredient.

15. Mixing the trunks and rootlets of ginseng (Panax ginseng C.A. Meyer) to prepare a ginseng mixture; preparing a primary extract using the mixed ginseng mixture as a primary solvent; filtering the primary extract and extracting the residue with a secondary solvent to prepare a secondary extract; Filtration of the secondary extract and extraction of the residue with a third solvent to prepare a third extract; and and mixing and concentrating the primary extract, the secondary extract, and the tertiary extract.

16. The method according to claim 15, wherein the ginseng mixture is a mixture of ginseng trunks and rootlets in a weight ratio of 3-9:1-7.

17. The method according to claim 15, wherein the ginseng multiple extract contains ginsenosides Rg1, Rf and Rb.

18. The method according to claim 17, wherein the ginsenosides Rg1, Rf and Rb are contained in a weight ratio of 8-10:4-6:18.5-22.

5.

19. mixing the trunks and rootlets of Korean ginseng or American ginseng to produce a ginseng mixture; Extracting the ginseng mixture to produce an extract; and and analyzing the extract using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

20. The method according to claim 19, wherein the trunks and rootlets are mixed in a weight ratio of 3-9:1-7.

21. 20. The method of claim 19, wherein in the ultra high performance liquid chromatography-quadrupole time-of-flight mass spectrometer, liquid chromatography is performed using 0.1% formic acid in water (v / v) and 0.1% formic acid in acetonitrile (v / v) as mobile phases and gradient elution.

22. 20. The method according to claim 19, wherein mass spectrometry is performed using an anion electrospray mode in the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer.

23. The method according to claim 19, wherein the origin of the Korean ginseng and American ginseng is determined by analyzing the major metabolites of the sample using an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer (UPLC-QTOF / MS) and performing multivariate statistical analysis on the types and amounts of the metabolites.

24. 20. The method of claim 19, wherein the multivariate statistical analysis is a multivariate statistical analysis using data from principal component analysis (PCA) or orthogonal partial least squares discriminant analysis (OPLS-DA).

25. 20. The method of claim 19, wherein the ginseng contains a metabolite selected from the group consisting of ginsenoside Rf, Rb2, malonyl-Rb2, Rg1, malonyl-Rc, 20-O-glucoginsenoside Rf, Rg2, and Rc.

26. 20. The method of claim 19, wherein the American ginseng contains a metabolite selected from the group consisting of ginsenosides Gypenoside XVII, Re, malonyl-Rb1, pseudo-ginsenoside F11 and Rb1.

27. Extracting Korean ginseng or American ginseng rootlets to produce an extract; and and analyzing the extract using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

28. 28. The method of claim 27, wherein the ginseng contains a metabolite selected from the group consisting of 20-O-glucoginsenoside Rf, Ra2, Ra1, Rf, Notoginsenoside R2, Rh1, and Rg1.

29. 28. The method of claim 27, wherein the American ginseng contains a metabolite selected from the group consisting of vinaginsenoside R4, notoginsenoside R1, notoginsenoside Fe, Re, compound O, and pseudo-ginsenoside F11 and F2.

30. administering to an individual a pharmaceutically effective amount of a mixed extract of the trunk and rootlets of ginseng (Panax ginseng C.A. Meyer).

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